Previous analysis of molecular chimeras between the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) and the v-ras transformation gene from Harvey murine sarcoma virus (HaMuSV) localized the steroid hormone regulatory sequences between 100 and 200 nucleotides upstream from the cap site in the LTR. This test system has been transferred to the single-strand DNA virus, M13, to permit high-resolution mutational analysis of the hormone regulatory signals. It has been found that the assay (hormone dependent appearance of v-ras dependent foci) remains functional in the new vector environment. In fact, the extent of hormone-dependence is even higher in the new system. A new method has been developed for the efficient and accurate introduction of mutations into regions of interest. This technique permits the oligonucleotide-directed introduction of mismatches as large as 10 nucleotides in a one-step procedure. The analysis of sequences required for the interaction of the hormone-receptor complex with target sequences in the DNA, and for the subsequent transcriptional response are in progress.